bioRxiv preprint doi: https://doi.org/10.1101/2020.05.13.093179; this version posted May 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Anaplerotic pathways in Halomonas elongata: the role of the sodium gradient Karina Hobmeier1;∗ Marie C. Go¨ess1;∗ Christiana Sehr1 Hans J¨orgKunte2 Andreas Kremling1 Katharina Pfl¨uger-Grau1 Alberto Marin-Sanguino1 May 13, 2020 * These two authors contributed equally to the current work. Affiliations: 1 Systems Biotechnology, Technical University of Munich, 85748 Garching, Germany 2 Department for Materials and Environment, BAM Federal Institute for Materials Research and Testing, Berlin Germany. Abstract Salt tolerance in the γ-proteobacterium Halomonas elongata is linked to its ability to pro- duce the compatible solute ectoine. The metabolism of ectoine production is of great interest since it can shed light on the biochemical basis of halotolerance as well as pave the way for the improvement of the biotechnological production of such compatible solute. The ectoine production pathway uses oxaloacetate as a precursor, thereby connecting ectoine production to the anaplerotic reactions that refill carbon into the TCA cycle. This places a high demand on these reactions and creates the need to regulate them not only in response to growth but also in response to extracellular salt concentration. In this work we combine modeling and ex- periments to analyze how these different needs shape the anaplerotic reactions in H. elongata. First, the stoichiometric and thermodynamic factors that condition the flux distributions are analyzed, then the optimal patterns of operation for oxaloacetate production are calculated. Finally, the phenotype of two deletion mutants lacking potentially relevant anaplerotic en- zymes: Phosphoenolpyruvate carboxylase (Ppc) and Oxaloacetate decarboxylase (Oad) is experimentally characterized. The results show that the anaplerotic reactions in H. elongata are indeed subject to different evolutionary pressures than those of other gram-negative bac- teria. Ectoine producing halophiles must meet a higher metabolic demand for oxaloacetate and the reliance of many marine bacteria on the Entner-Doudoroff pathway compromises the anaplerotic efficiency of Ppc, which is usually one of the main enzymes fulfilling this role. The anaplerotic flux in H. elongata is contributed not only by Ppc but also by Oad, an enzyme that has not yet been shown to play this role in vivo. Ppc is necessary for H. elongata to grow normally at low salt concentrations but it is not required to achieve near maximal growth rates as long as there is a steep sodium gradient. On the other hand, the lack of Oad presents serious difficulties to grow at high salt concentrations. This points to a shared role of these two enzymes in guaranteeing the supply of OAA for biosynthetic reactions. 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.13.093179; this version posted May 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Introduction The halophilic γ-proteobacterium Halomonas elongata DSM 2581T has a broad salt tolerance and can even grow in salt saturated brines (> 30% NaCl) [35] thanks to the accumulation of the compatible solute ectoine, which can reach molar concentrations in the cytoplasm without disrupting cellular processes. The synthesis of ectoine from its precursor oxaloacetate (OAA) withdraws a high amount of carbon out of the TCA cycle, which must then be replenished by anaplerotic reactions. This makes the PEP-Pyr-OAA node, already acknowledged as a major switching point for carbon metabolism [29], even more important for this organism. Figure 1: Anaplerotic reactions and TCA cycle in Halomonas elongata. Shown are the four enzymes PEP carboxylase (PPC, blue), Oxaloacetate decarboxylase (OAD, red), Malic enzyme (MAE, green) and Phosphoenolpyruvate carboxykinase (PCK, yellow) that have the potential to catalyze anaplerotic reactions Figure 1 shows the PEP-Pyr-OAA node and the glyoxylate shunt in H. elongata as it has been determined by genomic analysis [30, 26]. The glyoxylate shunt bypasses the two decarboxylation steps of the TCA cycle and thus enables the replenishment of C4 carbon skeletons from Acetyl coenzyme-A. This pathway allows an efficient growth on acetate but its operation on glucose as a carbon source would result in a loss of one third of the total carbon in the decarboxylation of pyruvate to Acetyl-CoA by Pyruvate DH. Growth on hexoses can be carried out efficiently thanks to the anaplerotic reactions. There are four enzymes that fullfil the stoichiometric requirements to catalyze anaplerotic reac- tions: PEP carboxylase (Ppc), Malic enzyme (Mae), PEP carboxykinase (Pck) and the membrane bound Oxaloacetate decarboxylase (Oad). The first of these enzymes, Ppc, has been known for a long time to carry most of the anaplerotic flux in Escherichia coli growing on glucose, glycerol or pyruvate [3, 24] and its regulation has been shown to play a critical role in carbon catabolism [37]. The second enzyme in the list, the malic enzyme (Mae), is present in two variants with different cofactor specificities (NADH and NADPH). The third candidate, phosphoenolpyruvate carboxykinase (Pck), is also an important enzyme in this node and catalyzes a key step for glu- coneogenesis converting of OAA into PEP. Finally, Oad deserves special attention in H. elongata. This enzyme catalyzes the interconversion of OAA into pyruvate. Cytoplasmic versions of this enzyme are considered to be irreversible, thermodynamically unable to function in the anaplerotic sense and usually involved in gluconeogenesis [25]. In H. elongata, however, Oad is a membrane 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.05.13.093179; this version posted May 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bound sodium pump [9] and it has been hypothesized [17] that it can operate as an anaplerotic reaction, thus using the considerable sodium gradient available under high salt concentrations to drive the carboxylation of pyruvate in H. elongata. To the extent of our knowledge, such a function has never been observed in vivo. The different physiological roles described so far for Oad go in the opposite direction, for example, as a sodium-transport system energized by the decarboxylation of OAA into pyruvate [8, 10, 6, 7] and as part of a catabolic pathway in a mutant strain of Lactococcus lactis [28]. The aim of this work is to shed light on the way anaplerotic reactions are organized in H. elon- gata through a combination of theoretical and experimental methods. Due to their mechanisms, the action of two of these enzymes (Oad and Ppc) cannot be distinguished through labeling exper- iments [23]. Therefore, we constructed mutant strains lacking each of the enzymes (Ppc and Oad) and characterized their phenotypes in comparison to the wild type. The results of this experiments are examined under the light of thermodynamic analysis and simulations of constraint based mod- els that are customized to deal with the challenge of estimating physico-chemical magnitudes at high salt concentrations. 2 Material and Methods Strains, plasmids, and growth conditions. H. elongata DSM 2581T and the mutant strains were routinely grown in minimal medium MM63 or LB medium containing 1 M NaCl at 30 o C under shaking at 220 rpm. All Escherichia coli strains were grown in LB medium at 37 o C amended with the antibiotic necessary for maintenance of the plasmid. All plasmids and genetic manipulations were performed in E. coli DH5α or E. coli DH5α λ-pir. Construction of plasmids pSEVA ∆ppc::Sm and pSEVA ∆oad::Sm. Replacement of the ppc gene with aadA, encoding a Streptomycin resistance cassette was performed by adapting the method previously reported for Pseudomonas putida [20]. The plasmids were assembled by Gibson Assembly (NEB) according to the suppliers manual. Oligonucleotides used for the gene amplifi- cation are shown in table 1. A DNA fragment containing about 500 bp of the genomic sequence upstream of the target gene ( ppc or oad) followed by the sequence of the aadA gene, encoding the streptomycin resistance cassette, and about 500 bp of the chromosomal region downstream of the target gene, was inserted into pSEVA212S, carrying the R6K suicide origin of replication [19], which had been previously linearized with EcoRI. The plasmid was transformed [5] into E. coli DH5α λ-pir and clones carrying it were selected on LB agar plates supplemented with 200 µg/ml streptomycin (Sm 200). The correct assembly of pSEVA ∆ppc::Sm and pSEVA ∆oad::Sm was verified by isolation of the plasmids and sequencing. Conjugal transfer of plasmids into H. elongata by triparental mating Plasmids were transferred to H. elongata by a two step mating procedure. First, plasmid pSW-2 encoding the 1-SceI endonuclease [20] was transferred to H. elongata. Therefore, the donor strain E. coli DH5α λ-pir (pSW-2), the recipient strain H. elongata, and the helper strain E. coli HB101 (pRK600) were grown in LB medium, amended with the antibiotic necessary to assure plasmid maintenance (pSW-2: 10 µg/ml gentamycin and pRK600: 35 µg/ml chloramphenicol) until the stationary phase (over night). Optical densities at 600 nm were measured and 1 ml of the donor culture was mixed with the amount of recipient and helper culture to obtain OD ratios of 1:1:1 and 1:2:2 respectively.